This invention relates to development of a new strategy to synthesize biomolecules that can be used to form structure-specific metallated biomolecules. The method provides a facile method using xe2x80x94PH2 intermediates to strategically place phosphine groups at specific sites on biomolecules that will facilitate formation of well-defined complexes on the biomolecule with radiometals and transition metals. These new metallated biomolecules hold potential for applications in the chemical and biomedical fields.
The transition metal chemistry of phosphines is diverse, resulting in a myriad of coordination compounds. The wealth of available data on the coordination chemistry of rhenium has provided a strong impetus in extending the chemistry to its diagonally related congener technetium-99. In fact, the demonstration that phosphine ligands produce well-defined, in vitro/in vivo stable complexes with technetium-99m (a xcex3-emitter with 141 keV and t1/2=6.2 h) has resulted in the development of two Tc-99m-based radiopharmaceuticals being currently used for myocardial imaging in humans. [DeRosch et al., 1992; Forster et al., 1992]. Because most human cancer cells express certain affinity for biomolecular vectors such as peptides or proteins, it is conceivable that radiolabeled receptor-avid peptides will provide new vehicles for delivering diagnostic or therapeutic radiations in site-directed treatments. Radiolabeling of receptor-avid peptides (or other bimolecular vectors) is best carried out by using bifunctional chelating agents. Radiolabeling with specific radioisotopes is done at the ligating unit of the bifunctional chelating agent, while functionalities such as xe2x80x94COOH or xe2x80x94NCS will incorporate a biomolecular vector within the bifunctional chelating agent to ultimately produce radiolabeled biomolecules. In this context, the utility of phosphines to construct new bifunctional chelating agents is attractive because of the potential applications of these ligands to produce well-defined complexes with radio-isotopes of diagnostic (Tc-99m) and therapeutic (Re-188, Au-199, Rh-105) value.
However, it may be recognized that chemical transformations of traditional phosphine ligands (e.g. Ph2PCH2CH2PPh2, dppe or Me2PCH2CH2Pme2, dmpe) into bifunctional chelating agents is a challenge. Aryl phosphines (e.g. dppe) which are oxidatively stable are unsuitable for use under in vivo conditions because of their high lipophilicty. On the other hand, alkyl phosphines are oxidatively so unstable that backbone modification and their use in aqueous media would produce corresponding phosphine oxides. Therefore, in order to utilize the superior ligating properties of phosphine ligands in the construction of new bifunctional chelating agents, new strategies on the overall design of phosphine frameworks were needed.
The rich chemistry of phosphines with transition metals makes them well suited for constructing chelating frameworks on simple and complex molecular structures that can be used to form well-defined metallated biomolecules. Metallated biomolecules, where the metal is bound (chelated) in a site-specific and structure-specific manner, hold important potential for a variety of chemical and biomedical applications, including chiral catalysis and radiopharmaceuticals [Gilbertson et al., 1996; Liu et al., 1997; Lister-James, et al., 1996]. In this context, the utility of phosphines to construct metal chelating frameworks either appended to or incorporated within biomolecular structures at specific positions is particularly attractive.
However, it must be recognized that the incorporation of phosphine functionalities in biomolecules by current synthetic strategies is challenging and usually involves lengthy procedures and harsh reaction conditions that often damage (e.g., reduction with Raney nickel) the biomolecule [Gilbertson et al., 1994]. For example, Gilbertson, et al. 1994, employed a reaction pathway to append diphenylphosphine groups that used a diphenylphosphorous (V) sulfide intermediate. After the P=S derivatized peptide was made, reduction of the P=S to the phosphorous (III) phosphine was accomplished with Raney Ni [3] producing a mixture of products where the desired diphosphine-peptide product was produced in low yields. The resulting diphenylphosphine-peptide conjugate was subsequently used to selectively form the corresponding Rh(III) conjugate [Gilbertson et al., 1994].
Recent efforts have been successful in synthesis of bidentate and multidentate chelation frameworks that contain di-hydroxymethylenephosphine (HMP) functionalities [i.e., xe2x80x94P(CH2OH)2] to facilitate formation of new transition metal complexes [Smith and Reddy et al., 1997; Smith and Katti et al, 1997; Smith and Li et al., 1997]. As a result of this work, the first bifunctional chelating agent containing HMP groups was synthesized, characterized, and used as a vehicle to conjugate metals to biomolecules. The synthesis of this bifunctional chelating agent system (i.e., carboxylate derivative of the di-HMP-di-thia (P2S2) tetradentate ligand framework shown in Formula 1) was difficult and proceeded via a xe2x80x94P(V)=0 intermediate (similar to the Gillickson xe2x80x94P(V)=S intermediate) that had to be reduced with LiA1H4 to a xe2x80x94P(III)H2 intermediate in route to formation of the xe2x80x94P(CH2OH)2 groups. However, the reduction conditions used would irreversibly alter most biomolecules precluding this approach for synthesis of most phosphine bioconjugates.
To overcome these difficulties, the inventors of the subject inventive method and product have realized and developed the use of xe2x80x94PH2 synthons to form xe2x80x94P(CH2OH)2 based ligands. Initially, a Brxe2x80x94(Ch2)3PH2 reactant was synthesized which demonstrated that it was possible to produce the P2S2-bifunctional chelating agent: 
P2S2xe2x80x94COOH bifunctional chelating agent (P2S2-BFCA) containing two HMP groups (R=xe2x80x94CH2OH).
This was done without going through the reduction step late in the synthetic scheme (see Schemes 1 and 2). It was unexpected that the utilization of the Br(CH2)3PH2 intermediate in this fashion would be facile given that phosphorous hydrides (e.g., xe2x80x94PH2 containing compounds) are known to have poor oxidative stability. However, these initial syntheses were carried out in organic solvents and care was taken to remove molecular oxygen, except for the rapid conversion of the xe2x80x94PH2 groups to the xe2x80x94P(CH2OH)2 groups by addition of water containing traces of H2CO.
The potential of using HMP-based chelating frameworks for labeling biomolecules with radioactive transition metals (including 99mTc and 188Re) has now been more fully explored. Recent studies of the current inventors demonstrate that 99mTc- and Re-complexes with the P2S2xe2x80x94COOH bifunctinal chelating agent (Formula 1) can be covalently linked to peptides to form well defined metal conjugates that have excellent in vitro and in vivo stability. These data provide important evidence that HMP-based metal chelates are useful systems for conjugating biomolecules with transition metals for a variety of chemical and biological applications. The inventors"" work with 99mTC- and Re-P2S2-bioconjugates and other model HMP-based chemical systems demonstrates that HMP-containing ligand frameworks hold important potential for the formulation of new diagnostic and therapeutic radiopharmaceuticals.
Alkyl substituted phosphines are, in general, oxidatively unstable and, therefore, their backbone chemical modification is difficult especially in the context of appending them on biomolecules (e.g. peptides, proteins and other receptor-avid biomolecules). The current discovery demonstrates that phosphines in the form of functionalized phosphorus(III) hydrides are oxidatively-stable and can be used to achieve chemical modifications. In fact, chemical backbones such as thioethers, amides and amines can be incorporated across xe2x80x94PH2 units without fear of oxidation of the PIII center. Further, the carboxylate functionalized (PH2)2S2xe2x80x94COOH bifunctional chelating agent is stable to reaction conditions that are employed during solid phase peptide synthesis (including treatment with HBTU, piperidine, solutions in DMF and washing the resin with trifluoroacetic acid of specific concentrations) for its incorporation on specific biomolecules including peptides (see Schemes 4 and 5). This order of chemical flexibility and oxidative stability of functionalized PIII hydrides present realistic prospects in the design and development of radiolabeled biomolecules for use in site-directed diagnosis and therapy of human cancers.